ProjectSummary/Abstract Hirschsprung?s disease (HSCR) is a common cause of neonatal bowel obstruction that results from failed development of the distal enteric nervous system (ENS). The pathophysiology, approach to diagnosis and surgicaltreatmentofHSCRhasbeenwell-definedoverthelast60years.However,Hirschsprung?s-associated enterocolitis (HAEC), the most serious complication of HSCR, remains a frequent cause of pre- and post- operative morbidity and mortality in HSCR patients, with poorly defined pathophysiology and unchanged treatmentguidelinesovermultipledecades.HAECisthepresentingsymptomofHSCRin25%ofinfantsand mortalityrangesfrom1-10%,withthemajorityofdeathsoccurringinnewbornspriortodefinitiveoperation.Our centralhypothesisisthatdevelopmentaldeficiencyoftheENSinHSCRisaccompaniedbydefectsinintestinal homeostasis,leadingtoincreasedsusceptibilitytoHAEC.Advancesinunderstandingthegeneticsunderlying HSCRhavealloweddevelopmentofanimalmodelswhichrecapitulatethehumanHAECphenotype.Overthe last five years, studies from our laboratory utilizing the EdnrBNCC-/- model of HSCR/HAEC have allowed us to identify defects in intestinal homeostasis that contribute to HAEC. We have identified ENS alterations in the ?normal?, ganglionated colon of EdnrBNCC-/- mice which include decreased neuronal density and a shift in neurotransmitterphenotypes,withover-representationofrelaxationneurotransmittersandunder-representation of contractility neurotransmitters. Additionally, we have demonstrated that EdnrBNCC-/- mice develop colonic dysbiosispriortoHAEC.Mostrecently,preliminarydataindicatealterationsincolonicepithelialtightjunctions precedingthedevelopmentofHAEC.Furthermore,EdnrBNCC-/-intestinalsegmentshaveincreasedsusceptibility toinvasionbypathogenicE.coli,withdecreasedproductionofinnateimmunedefensemolecules.Finally,we have identified impaired cellular immunity (B-cell populations and decreased secretory IgA) in these animals. Takentogether,intestinalhomeostasisinEdnrBNCC-/-miceappearstogovernedbyafour-wayinterplaybetween theentericnervoussystem,luminalmicrobiota,theintestinalepithelialbarrier,andthemucosalimmunesystem, allofwhichappeartobeperturbedinthesettingofalteredhostgenetics.However,ourstudiestodatehavenot allowedustodistinguishwhichalterationsinintestinalhomeostasisarecausativeversusassociatedwithHAEC. Recently, intestinal enteroids have emerged as a tool to model the complexity of the intestinal epithelium in culture,avoidingconfoundinginfluencefromlocalandsystemnon-epithelialsources.Inordertoadvanceour understanding of the complex dynamics that contribute to HAEC, we propose to develop an experimental paradigm of in vivo, ex vivo and in vitro methods that will allow combinatorial testing of the components of intestinalhomeostasis.ThisproposalwillevaluatethecontributionsofEdnrBNCC-/-microbiomedysbiosisand epithelialbarrierdysfunctiontothedevelopmentofHAECandwilldeterminethesuitabilityofintestinalenteroids asanovelsystemtostudythepathogenesisofHAEC.